Abstract: In humans, clinically relevant disease with Toxoplasma gondii, a common intracellular parasite, results from T.gondii?s tropism for and life-long persistence in the CNS. To persist, T.gondii switches from a fast replicating form (tachyzoite) to a slow replicating, encysted form (bradyzoite). While T.gondii infection is asymptomatic in most, T. gondii can cause severe neurologic complications in individuals with incomplete immune responses (e.g. AIDS patients, developing fetuses) including cognitive, visual and motor deficits, and death. The treatments for symptomatic toxoplasmosis, most commonly pyrimethamine and sulfadiazine, are poorly tolerated, have low therapeutic indices, and are ineffective against the persistent, encysted form. The development of therapies targeting T. gondii?s encysted form requires a deep, mechanistic understanding of how parasites encyst. Prior studies have focused on identifying parasite genes that drive the tachyzoite/bradyzoite transition and parasite proteins that form the cyst wall. Less work has been done on identifying host cell genes that influence encystment, and almost no work has been done on how parasite manipulations of the host cell affect encystment. The goal of this proposal is to address this gap by building upon my preliminary work showing that a well-known kinase (ROP16)? which parasites injects into the cytoplasm of host cells and which shows allelic variation between the canonical T. gondii strains (type I, II, and III)? facilitates encystment in a strain-specific manner. Type I and III alleles (rop16I/III/ROP16I/III), which are 99% identical, cause prolonged activation of STAT3, 6, and possibly 5, signaling pathways during acute tachyzoite infection, while the type II allele does not. To determine if ROP16 play a role in encystment, I generated type II and type III strains that lack ROP16 (II?rop16 and III?rop16) and tested them in an in vitro encystment assay. Remarkably, in contrast to II?rop16 which showed little change in encystment, III?rop16 has a greater than 2-fold decrease in forming cysts in fibroblasts and neurons. In addition, I found that this defect can be complemented in trans by co-infecting a host cell with III?rop16 and parental type III (WTIII) parasites, but not with type II parasites, suggesting a role for strain-specific host cell manipulations. My overall hypothesis, therefore, is that ROP16III facilitates cyst development through strain-specific host cell manipulations. I will address this hypothesis by: i) determining which ROP16III functions are required for encystment by complementing the III?rop16 strain with a panel of ROP16 mutants that lack kinase activity, nuclear localization, or STAT-binding and assessing encystment (Aim 1) and ii) identifying the ROP16III-dependent host genes that drive encystment by transcriptionally profiling cells in encysted conditions and infected with WTIII, III?rop16, or complemented parasites (Aim 2). The completion of this work will significantly advance our understanding of how T.gondii encystment varies by strain type, a necessary first step toward developing strain-specific drugs against the encysted form of T. gondii.